Category: Formulation and Quality
Purpose: Calcium phosphate nanoparticles (CaPs) , loaded with the mimetic peptide (MP), selectively targeting the cytosolic Cavβ2 chaperon subunit of the cardiac L-type Calcium Channel Complex (MP), showed a significant activity after intratracheal insufflation in rats . MP is a novel therapeutic substance able to recover altered myocardial functions. The nanoparticle embedded in an inhalable powder would allow a lung driven administration. The goal of this first step research was to design and develop by spray drying (SD) a highly respirable microparticulate dry powder (dpCaPs) able to embed and release the unloaded CaPs. The polyalcohol mannitol was selected as a soluble, size protective and safe carrier for microparticle construction . The microparticles carrying nanoparticles have to be inhaled and deposited into alveoli; the embedded CaPs, restored by release from microparticulate carrier dissolution, can be translocated across gas-blood barrier for direct transport to heart.
A Design of Experiment was applied for understanding the effect of the dispersion composition and spray drying process parameters on selected critical quality attributes of the microparticles embedding CaPs, with the objective to optimize the scale up process.
Methods: An aqueous solution of CaCl2 0.1M with Na3Cit and a Na2HPO40.12M solution were mixed at pH 9.0. The precipitated nanoparticle dispersion was kept at 25°C for 24 hours and then, centrifuged to remove unreacted salts. The final concentration of nanoparticles was 7.0 mg/ml. To construct inhalable microparticles by spray drying (Büchi B-290, CH), mannitol was dissolved in nanoparticle dispersion at the selected mannitol percent. The fixed SD parameters were: Inlet temperature 125°C, Aspiration rate 35 m3/h and nozzle diameter 0.7 mm. Powder morphology was characterized by FIB-SEM.The micro and nanoparticle size distribution were measured by laser and dynamic light scattering, respectively. Residual solvent was assessed by TGAanalysis;the mannitol polymorphism was investigated by DSC. In vitro respirability was evaluated usinga medium resistance RS01®(Plastiape,IT)inhaler at 60L/min, using the Fast Screening impactor (Copley Scientific, UK).
The Cubic Central Composite Experimental Design applied required to run eleven experiments i.e., eight experiments for the full factorial design (two levels per factor: 23) and three experiments as central points for the design statistics. Feed rate (3.5 - 7.0 ml/min), mannitol/CaPs ratio (50 - 80%) and CaPs concentration (0.5 - 7.0 mg/ml) were the three composition or process parameters fixed at two levels equally distant from the central point. For the evaluation of the product performance, the multivariate analysis was conducted by Design-Expert® software. Eleven experimental powders were manufactured according to the Central Composite Design. Yield of SD, median volume diameter, residual solvent, Emitted and Fine Particle Dose of dpCaPs were elected as Critical Quality Attributes, together with the size of the restored CaPs in aqueous medium.
Results: Unloaded dpCaPs morphology revealed spherical (Fig.A) and doughnut structures (Fig.B), function of the nanoparticles and mannitol concentration in the dispersion solution to spray dry. CaPs in the mannitol matrix resulted embedded on the surface of microparticles; this peculiar morphology is favourable to CaPs release during microparticle dissolution in the lung fluid. From DSC analysis, the mannitol fusion peak indicated the presence of stable bform of polyalcohol. The dpCaPs respirability, as Emitted Dose and Fine Particle Dose, was between 32.1 and 38.1 mg and 11.8 to 24.4 mg, respectively, in front of 40 mg of powder loaded. The DoE study allowed to identify the quality attributes significantly affected by the process and composition parameters (p< 0.05). Namely, the increase of CaPs dispersion feed rate during drying process negatively affects all powder quality attributes.
Conclusion: To SD manufacture dpCaPs with high yield, good aerodynamic performance and small size of restored CaPs, the percentage of added mannitol respect to the CaPs has to be higher than 65%. Decreasing the CaPs concentration in the dispersion, an increase of fine particle dose and CaPs nanosize after their restoration was measured; on the contrary, emitted dose and SD yield resulted diminished.
 PCT/EP2015/080991 and Di Mauro et al. Nanomedicine 2016, 8:891-906
 Rusconi et al. Circulation 2016, 134:534–546; Miragoli et al., Science Translational Medicine 2018, 10,424,eaan6205
 A. Torge et al, European Journal of Pharmaceutical Sciences 2017, 104:171-179